PROJECT SUMMARY Candidate Caroline Sokol, M.D., Ph.D., is an Assistant in Medicine in the Allergy and Clinical Immunology Unit at Massachusetts General Hospital (MGH) and an Instructor of Medicine at Harvard Medical School (HMS). She received her M.D./Ph.D. from Yale University, where she studied the mechanisms of innate immune control of Th2 differentiation in the laboratory of Dr. Ruslan Medzhitov. Her work there led to two first author Nature Immunology publications on the role of basophils in initiating the allergic immune response and Th2 differentiation. It also led to her current work in the laboratory of Dr. Andrew Luster at the Center for Immunology and Inflammatory Diseases (CIID) at MGH, where she has focused on the role of innate immune cell trafficking in the activation and control of the allergic immune response and Th2 differentiation. The short- term goals of this K08 award application resubmission are to provide training in intravital microscopy, advanced genomic techniques, including RNA-Seq, human immunology and translational research. Dr. Sokol?s long-term goal is to develop an independent translational research program studying the innate immune control of allergic disease. The experiments, training activities, and mentoring plan outlined in this proposal will successfully position Dr. Sokol for her first R01 and an independent career as a physician-scientist. Mentorship, Training Activities, and Environment Dr. Sokol will perform the work outlined in this proposal in the CIID at MGH, under the mentorship of Dr. Andrew Luster. The CIID is a state-of-the-art multidisciplinary basic science research center focused on mechanisms of immune-mediated inflammatory diseases that serves as the foundation for immunology research at MGH. Dr. Luster is an expert in the fields of cellular trafficking and chemokine research, areas that are central to this application. Additionally, Dr. Luster has an excellent track record of mentorship, having mentored over 50 post-doctoral candidates, of which 17 are currently independently-funded research faculty at academic institutions. Additionally, Dr. Sokol has organized a Training Advisory Committee consisting of Drs. Nir Hacohen, Thorsten Mempel and Arlene Sharpe. They will provide expertise and hands-on training in advanced microscopic imaging, advanced genomic techniques, human immunology and translational research. To complement the expertise of her mentors, Dr. Sokol will complete didactic courses in bioinformatics and systems biology, grant writing, leadership, translational research, and the responsible conduct of research through the Harvard Catalyst. The collaborative opportunities, intellectual environment, and resources available to Dr. Sokol are outstanding and provide her with a clear path to independent success. Research Allergic diseases are characterized by inappropriate Type-2 immune responses targeted against non-infectious environmental allergens. Despite the high prevalence of allergic diseases, very little is known about how the innate immune system recognizes allergens and initiates the allergic immune response, which has limited research into novel therapeutics. Recent research has identified a specialized population of CD301b+ dendritic cells (DCs) located in murine skin that selectively migrates into the draining lymph node (dLN) and promotes Th2 differentiation in response to allergens. However, the pathways that promote this selective migration in response to allergens are unknown. We propose first, to define the chemokine pathways involved in the migration of CD301b+ DCs to the dLN. We show in preliminary data that allergen exposure results in the production of CCR8 ligands in the skin and dLN of mice, as well as in human skin explants. Furthermore, we provide preliminary data that in addition to CCR7 signals, CCR8 is necessary for the emigration of CD301b+ DCs from the skin and subsequent Th2 differentiation, while CCR2 prevents CD301b+ DC emigration to the dLN. However, control of CD301b+ DC migration is not the only mechanism for innate control of allergic immune initiation. Although CD301b+ DCs are necessary for Th2 differentiation, they are not sufficient to induce Th2 differentiation from nave T cells. We next propose to determine the role and identity of accessory cells in promoting Th2 differentiation. We provide preliminary data that there is a required accessory cell that provides the necessary skewing information in tandem with antigen presentation and costimulation by DCs. These accessory cells enter the dLN via a CD62L-dependent process and the entry of this cell type corresponds with the entry of a previously undescribed IL-4 producing cell that we have identified. We believe that through the control of CD301b+ DC migration and the entry of this accessory cell, that the innate immune system precisely controls the outcome of T helper cell differentiation.